7. Project Summary/Abstract Liver metabolism and gene expression show striking sex differences regulated by the sex-specific temporal patterns of pituitary growth hormone (GH) secretion and GH-stimulated activation of STAT5, both of which are pulsatile in males and persistent in females. The long-term goal of this project is to elucidate sex differences in the liver transcriptome, which are widespread in both mice and humans, to better understand the transcriptional underpinnings of the clinically relevant sex differences in hepatic drug and steroid metabolism, lipid metabolic profiles, fatty liver disease, and liver fibrosis and hepatocellular carcinoma development. Advances during the last project period revealed that GH establishes a sex-differential epigenomic environment that enables its sex-specific actions in the liver. Several thousand putative enhancers with sex-biased binding sites for STAT5 and other essential GH-regulated liver transcription factors were identified, and sex-specific deposition by Ezh1/Ezh2 of histone H3- lysine 27 trimethylation (H3K27me3) was shown to be required for repressing many female-biased genes in male liver. Further, more than 200 sex-specific, GH-regulated and nuclear-enriched long, non-coding RNAs (lncRNAs) were discovered, and candidates for regulation of the sex-differential deposition of H3K27me3 and other chromatin marks at sex-specific genes and their enhancers were identified. Fundamental questions raised by these findings include: 1) what determines whether a given sex-biased enhancer is functional at activating gene transcription in a sex-biased manner; 2) how do active sex-biased enhancers select their distal sex-biased gene targets; and 3) how do the sexually dimorphic plasma GH patterns establish and maintain the sex-biased chromatin states that are functionally linked to the expression of sex-biased genes impacting liver physiology and disease susceptibility. This project uses the mouse model to address these questions and elucidate the essential biological mechanisms that underlie the complex transcriptional and epigenetic regulation by GH of liver sex differences and hepatic function. The work proposed has two major aims: 1) to uncover the design principles that underpin sex-biased gene transcription by identifying functionally active sex-biased enhancers and by elucidating their organization with sex- biased target genes within chromatin loop domains and subdomains; and 2) to discover the role of sex-specific, GH- regulated lncRNAs in establishing and maintaining the sex-differentiated chromatin states that support sex differences in liver gene transcription. Together, these studies will identify the central mechanistic features that enable GH, and its sex-dependent plasma patterns, to regulate the sex-biased expression of hundreds of genes that control key liver metabolic processes having a major impact on human health and liver disease, and may link molecular features to physiological and pathophysiological outcomes. The results obtained will have a high impact on research in this field by shifting the mechanistic focus of genomic and epigenomic studies of GH action from correlation and inferred function to causality, and will serve as a paradigm for studies of pulsatile hormone action for other endocrine factors that act through complex epigenetic mechanisms. !